Amino-acyl-type and catecholamine-type contrast agents for MRI

ABSTRACT

The present invention relates to the preparation of amino-acyl-type and catecholamine-type compounds having multiple carboxylic acid functional groups. Paramagnetic metal (II) or (III) ion chelate complexes are formed using these compounds for use as intravenous contrast agents to produce enhanced contrast magnetic resonance images of the heart, liver, biliary tree or upper small intestine. The mono- and di-amino acids, their esters and amides, and catecholamine-like derivatives, of EDTA, DTPA, and the like are prepared. The paramagnetic metal (II) or (III) ion complexes are formed and produce T1-related contrast effects in MR images. The compounds and complexes also appear to have low toxicities and to be relatively rapidly and completely cleared from the tissue of a living mammal, e.g. a human being.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 08/169,301filed Dec. 17, 1993 U.S. Pat. No. 5,562,894 which is acontinuation-in-part of U.S. Ser. No. 08/086,349, filed Jun. 30, 1993(abandoned); which is a contuation-in-part of U.S. Ser. No. 07/927,172,filed Aug. 7, 1992 (abandoned); which is a contuation-in-part of U.S.Ser. No. 07/744,470, filed Aug. 12, 1991 (abandoned); which is acontuation-in-part of U.S. Ser. No. 07/743,143, filed Aug. 9, 1991(abandoned).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the preparation and use ofamino-acyl-type and catecholamine-type hepatobiliary and cardiaccontrast agents useful in magnetic resonance imaging. The contrastagents have multiple carboxyl groups to chelate a variety of metal (II)or (III) ions.

2. Description of Related Art

This invention relates to contrast agents for medical magnetic resonanceimaging (MRI).

A contrast agent is an exogenous substance that either augments orsuppresses the normal in vivo MRI signal, thereby yielding additionaldiagnostic information. The theory and applications of various types ofcontrast agents have been described in the literature (1,2). The Arabicnumbers in parentheses in this section refer to the articles cited inthis section.!

The applications of a given MRI contrast agent are determined by itsdistribution in vivo. The mechanisms controlling the initialbiodistribution can be classed as physico-chemical, i.e., dependent onlyupon such properties as molecular size, charge, lipophilicity, surfaceproperties, etc.; or receptor-mediated, i.e., dependent upon the bindingof a substrate to a specific receptor in or on cells. Different organsmay handle the same contrast agent by different mechanisms. For example,the molecular size of the agent may result in its filtration by thekidneys (or confinement to the vascular space) while it is cleared byreceptor-mediated transport in the liver.

Contrast agents exhibiting a physico-chemical distribution mechanisminclude the gadolinium (III) complex of diethylenetriaminepentaaceticacid (Gd-DTPA), which distributes in blood plasma and extracellularfluid, and albumin-(Gd-DTPA)_(n), which remains largely intravascular(1,2). The former is used to demonstrate blood-brain barrier lesions orto reveal renal anatomy and function (3), while the latter has been usedexperimentally to delineate the vasculature (4) and determine brainblood volume (5,6). Iron-dextran, although a colloid, has a sufficientlylong plasma half-life (12 hr) to be used as an intravascular T2 contrastagent (7), as do some superparamagnetic iron oxide particle preparations(8,9).

Because of its role in the removal of exogenous compounds from generalcirculation, the liver is able to actively take up and concentratesoluble, as well as particulate, contrast agents. The pathways followedby solutes from plasma to bile have been reviewed (10-11) and arediagrammed in FIG. 4. Passage into the hepatocyte across the cellmembrane can take place by pinocytosis, passive diffusion, and/or bycarrier-mediated systems that transport bile acids, bilirubin, organicanions, organic cations, neutral organic compounds, or inorganic ions.The substrate specificity of different carrier systems can partiallyoverlap (e.g., organic anions and bile acids). The substrate may bemetabolized intracellularly and/or conjugated with glucuronic acid orglutathione, for example. Finally, excretion into bile canaliculi againinvolves passage through a cell membrane. The mechanism of biliaryexcretion for a given compound may differ from that operative for itsuptake.

The relative rates of metabolism, biliary elimination, and renalexcretion determine the clearance of drugs and their metabolites fromblood plasma and their persistence in any one organ system. However,presently the factors that direct one compound to be excreted in thebile and another in the urine are not completely understood. Molecularweight, polarity, and molecular structure in relation to binding toplasma and transporter proteins are important. There appears to be ageneral molecular weight threshold, which is species-dependent (ca. 300for rats and 600 for humans) below which urinary excretion dominates(10-11). Hydrophilic-lipophilic balance appears to play a critical rolein biliary excretion (10-11). However, a priori prediction is notpresently possible.

The liver has provided the first example of receptor-mediatedlocalization of an MR contrast agent--Fe-EHPG (EHPG isEthylene-bis(hydroxyphenylglycine)) (12). Other iron (13-15), manganese(16-17), and gadolinium (18) chelates have since been described thathave either potential for, or have demonstrated receptor-mediatedhepatocyte uptake.

It has been reported by others that the anionic chelates Fe-EHPG,Fe-HBED (HBED=bis-(hydroxybenzyl) ethylenediaminediacetic acid), andFe-PGDF (PGDF=N-3-(phenylglutaryl) desferrioxamine B) are transported inthe liver by a system or systems inhibitable by BSP(bromosulfophthalein) (13,15).

The lipophilic chelate Gd-BOPTA ("benzyloxypropionic-tetraacetate," aderivative of DTPA) was shown to have significant biliary excretion(38.6% of injected dose in bile at 6 hr) (18). No information wasreported on the mechanism of transport (e.g., passive diffusion oranionic transport) of this compound. Gd-BOPTA produced a larger signalenhancement (48%) in liver than Gd-DTPA (16%) in T1-weighted spin-echoimages at 0.5 Tesla.

Additionally, other organs and tissues may possess receptors withaffinity for certain classes of substrates, e.g., amino acids, peptidesor catechol amines (19-24). These receptors may also bind molecules thatresemble the substrate, e.g., a derivative of an amino acid that ispresent in a peptide substrate (22) or an amide derivative of anaturally occurring catechol amine such as dopamine. The contrast agentsof this invention may in part localize by such a mechanism. Furthermore,the localization of the catechol containing contrast agent of thepresent invention may depend in part on their respectivereduction-oxidation properties.

To date, magnetic resonance imaging (MRI) has played a minor role inimaging of the liver and abdomen of a human being because of degradationof image quality by motion artifacts, and by the lack of suitablecontrast agents. Recent technical advances in instrumentation (e.g.,self-shielded gradient coils) and pulse sequences (e.g., echo-planar andturbo-flash techniques) promise to alleviate the motion-related problemsof the torso and abdomen, and make contrast agent development all themore important for continued progress in abdominal MRI.

General background in the use of MRI contrast agents and of theirpreparation and purification are described, for example, in:

H. Gries et al., U.S. Pat. No. 4,647,447;

R. B. Lauffer et al., U.S. Pat. No. 4,899,755 and 4,880,008;

B. L. Engelstad et al., U.S. Pat. No. 4,909,257;

D. L. White et al., U.S. Pat. No. 4,999,445.

1. R. B. Lauffer, "Paramagnetic metal complexes as water protonrelaxation agents for NMR imaging: Theory and Design," Chem. Rev.(1987); 87:901-927.

2. S. M. Rocklage, et al. "Contrast Agents in Magnetic ResonanceImaging." Chapter 14, in Magnetic Resonance Imaging, 2nd ed., Stark D.D., Bradley W. G., eds. St. Louis: C. V. Mosby Co. (1992).

3. G. Bydder, "Clinical applications of Gadolinium-DTPA." in MagneticResonance Imaging. Stark D. D., Bradley W. G., eds. St. Louis: C. V.Mosby Co. (1988); 182-200 (Chap. 10).

4. M. E. Moseley et al., "Vascular mapping using Albumin-(Gd-DTPA), anintravascular MR contrast agent, and projection MR imaging," J. ComputerAssist Tomography (1988); 13:219-221.

5. T. A. Kent et al., "Cerebral blood volume in a rat model of cerebralischemia by MR imaging at 4.7 T,"AJNR (1989); 10:335-358.

6. D. L. White et al., "Determination of perfused cerebral blood volumeusing an intravascular MR contrast agent," Book of Abstracts: Society ofMagnetic Resonance in Medicine (1989); 2:806.

7D. L. White et al., "Iron-Dextran as a magnetic susceptibility contrastagent: Flow-related contrast effects in the T2-weighted spin-echo MRI ofnormal rat and cat brain," Magn.Reson.Med. (1992); 24:14-28.

8. D. L. White et al., "Plasma clearance of ferrosomes, a long-livedsuperparamagnetic MRI contrast agent." Book of Abstracts: Society ofMagnetic Resonance in Medicine (1990); 1:51.

9. R. Weissleder et al., "Ultrasmall superparamagnetic iron oxide:Characterization of a new class of contrast agent for MR imaging,"Radiology (1990); 175:489-493.

10. L. S. Schanker, "Secretion of organic compounds into bile." in TheHandbook of Physiology. Alimentary Canal V. Washington, D.C.: AmericanPhysiol. Society, Chap. 114:2433-2449.

11. C. D. Klaassen et al., "Mechanisms of bile formation, hepaticuptake, and biliary excretion," Pharm. Rev. (1984); 36:1-67.

12. R. B. Lauffer et al., "Iron-EHPG as a hepatobiliary MR contrastagent: Initial imaging and biodistribution studies," J. Computer Assist.Tomoaraph, (1985); 9:431-438.

13. B. Hoener et al., "Evaluation of Fe-HBED and Fe-EHPG as magneticresonance contrast agents for assessing hepatobiliary function," J.Magn. Reson. Imaging, (1991); 1:357-362.

14. K. A. Muetterties et al., "Ferrioxamine B derivatives ashepatobiliary contrast agents for magnetic resonance imaging," Magn.Reson. Med. (1991); Vol. 22, pp. 88 to 100.

15. B. Hoener et al., "Hepatic transport of the magnetic resonanceimaging contrast agent Fe(III)-N-(3-Phenyl-glutaryl) desferrioxamine B,"Magn. Reson. Med. (1990); 17:509-51.

16. D. L. White et al., "Clearance, excretion, and organ distribution ofa new MRI contrast agent Manganese-Dipyridoxal-Diphosphate (Mn-DPDP)."Abstract Book: Society of Magnetic Resonance in Medicine (1988) 1:531.

17. S. W. Young, "MRI measurement of hepatocyte toxicity using the newMRI contrast agent manganese dipyridoxal diphosphate, amanganese/pyridoxal 5-phosphate chelate," Mag. Reson. Med. (1989);10:1-13.

18. P. Pavone et al., "Comparison of Gd-BOPTA with Gd-DTPA in MRIimaging of rat liver," Radiology (1990); 176:61-64.

19. P. Ascher, "Glutamate receptors and glutamatergic synapses. InReceptors. Membrane Transport and Signal Transduction. A. E.Evangelopoulis et al., Berlin: Springer Verlag. (1989): 127-146.

20. F. P. Lehman, "Stereoselective Molecular Recognition in Biology. InReceptors and Recopnition, Vol. 5, Series A. Cuatrecasas P. and GreavesM. F. London: Chapman-Hall (1978).

21. R. D. O'Brien, ed. The Receptors, A Comprehensive Treatise, Vol. 1,New York: Plenum Press (1979).

22. S. S. Schiffman et al., "The Search for Receptor that MediateSweetness," In The Receptors, Vol. 4, Conn, P. M., ed. Academic Press.Orlando. (1986).

23. A. S. Horn, et al., eds. The Neurobiology of Dopamine. AcademicPress. New York. 1979.

24. B. J. Clark. "The role of dopamine in the periphery," in TheDopaminergic System, B. Halasz, et al., eds., Springer-Verlag, Berlin,1985, p 27-39.

All reference articles, patents, etc. cited in this application areincorporated herein by reference in their entirety.

It would be very useful to have organic chelate metal ion complexeswhich are specific for MRI imaging of the liver, the biliary tree, theupper small intestine, or the myocardial tissue. The present inventionprovides complexes and methods having these useful advantages.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to an amino-acyl-typemagnetic resonance imaging contrast agent, comprising the complex:

    L.sup.1 --M

wherein M is a metal (II) or (III) ion independently selected from thegroup consisting of metals of atomic number 21 to 31, metals of atomicnumber 39 to 50, the lanthanide metals having an atomic number from 57to 71, and metals of atomic number 72 to 82; and

L¹ is a polydentate amino-acyl-type chelating moiety of Formula 1:##STR1## wherein Q, J, X, X' and Z are each independently selected fromthe group consisting of --CH₂ --(C═O)OR¹ and --CH₂--(C═O)--NH--CH(R)(A);

wherein each R is independently selected from the group consisting of-hydrogen, --K, --W and --K--W, wherein each K is an alkyl group having1-7 carbon atoms, and each W is independently selected from the groupconsisting of aryl, substituted aryl, heteroaryl and substitutedheteroaryl; and

each A is a carbonyl-containing moiety independently selected from thegroup consisting of --(C═O)OR¹ and --(C═O)--N(R²)(R³), wherein R¹, R²and R³ are independently selected from the group consisting of hydrogen(i.e. the acid), alkyl having 1-7 carbon atoms, cyclohexyl, phenyl,benzyl, 1-naphthyl and 2-naphthyl, provided that when every A is--(C═O)OR¹ and every R¹ is hydrogen, then at least one R is --W or--K--W; and

m is selected from 0, 1, 2 or 3, and

n is selected from 0 or 1;

or the pharmaceutically acceptable salt(s) thereof.

Another aspect of this invention relates to a polydentateamino-acyl-type chelating compound (L¹) of Formula 1, supra.

Another aspect of this invention relates to a method of preparing apolydentate amino-acyl-type chelate compound (L¹) of Formula 1, whichmethod comprises:

(a) contacting a structure of the following formula: ##STR2## wherein Dand E are CH₂ (C═O)OR¹, with an amino acid of the structure H₂N--CH(R)--(C═O)OH, an ester of the structure H₂ N--CH(R)--(C═O)OR¹ or anamide of the structure H₂ N--CH(R)--(C═O)--N(R²)(R³);

wherein each R is independently selected from the group consisting of--K, --W and --K--W, wherein each K is an alkyl group having 1-7 carbonatoms, and each W is independently selected from the group consisting ofaryl, substituted aryl, heteroaryl and substituted heteroaryl;

R¹, R² and R³ are independently selected from the group consisting ofhydrogen (i.e. the acid), alkyl having 1-7 carbon atoms, cyclohexyl,phenyl, benzyl, 1-naphthyl and 2-naphthyl, and

m is selected from 0, 1, 2 or 3, and

n is selected from 0 or 1,

in an anhydrous dipolar aprotic solvent at between about 50 and 150°;and

(b) removing the solvent and recovering the compound of Formula 1.

Another aspect of this invention relates to a method of examining amammal in a diagnostic manner, which method comprises:

(a) injecting the mammal with an amino-acyl-type contrast agent in adose amount having a concentration of the complex L¹ --M of betweenabout 0.5 and 5000 micromol/kg of body weight of the mammal;

(b) placing the mammal of step (a) in a magnetic field irradiating withradio-frequency energy such that nuclear magnetic resonance can bedetected; and

(c) analyzing the imaging nuclear magnetic resonance signals obtained.

In another aspect, the present invention relates to a catecholamine-typemagnetic resonance imaging contrast agent, comprising the complex:

    L.sup.2 --M

wherein M is a metal (II) or (III) ion independently selected from thegroup consisting of metals of atomic number 21 to 31, metals of atomicnumber 39 to 50, the lanthanide metals having an atomic number from 57to 71, and metals of atomic number 72 to 82; and

L² is a polydentate catecholamine-type chelating moiety of Formula 2:##STR3## wherein G, T, V, V' and Y are each independently selected fromthe group consisting of --CH₂ --(C═O)OR¹ and --CH₂ --(C═O) --NH--CH₂--R⁴ ;

wherein each R⁴ is independently selected from the group consisting of--CH₂ -aryl, --CH₂ CH₂ -aryl, --CH₂ -(substituted aryl), and --CH₂ CH₂-(substituted aryl); and

m is selected from 0, 1, 2 or 3, and

n is selected from 0 or 1;

or the pharmaceutically acceptable salt(s) thereof.

Another aspect of this invention relates to a polydentatecatecholamine-type chelator (L²) of Formula 2, supra.

Another aspect of this invention relates to a method of preparing apolydentate catecholamine-type chelator (L²) of Formula 2, which methodcomprises:

(a) contacting a structure of the following formula: ##STR4## wherein Dand E are CH₂ (C═O)OR¹, with a derivative of the structure NH₂ --CH₂--R⁴ ; wherein R⁴ is independently selected from the group consisting of--CH₂ -aryl, --CH₂ CH₂ -aryl, --CH₂ -(substituted aryl), and --CH₂ CH₂-(substituted aryl); and

m is selected from 0, 1, 2 or 3, and

n is selected from 0 or 1,

in an anhydrous dipolar aprotic solvent at between about 50 and 150°;and

(b) removing the solvent and recovering the compound of Formula 2.

Another aspect of this invention relates to a method of examining amammal in a diagnostic manner, which method comprises:

(a) injecting the mammal with a catecholamine-type contrast agent in adose amount having a concentration of the complex L² --M of betweenabout 0.5 and 5000 micromol/kg of body weight of the mammal;

(b) placing the mammal of step (a) in a magnetic field irradiating withradio-frequency energy such that nuclear magnetic resonance can bedetected; and

(c) analyzing the imaging nuclear magnetic resonance signals obtained.

These metal ion chelates produce T1 contrast effects in the heart,liver, biliary tree, and upper small intestine. They demonstratefunction, as well as anatomy. These contrast agents have low toxicities,and unlike iron from superparamagnetic particulates, the metal fromthese compounds should be rapidly and relatively completely cleared fromthe body. Therefore, these contrast agents are of substantialsignificance to useful abdominal MRI.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C and 1D are each a representation of the structures ofthe compounds BOPTA, BSP, DPDP and DTPA, respectively.

FIGS. 2A, 2B, 2C and 2D are each a representation of the structures ofthe chelates EDTA, EDTP, EHPG and HBED, respectively.

FIG. 3 is a representation of a species of the general reaction toproduce a bis amino acid substituted chelate.

FIG. 4 is a cross-sectional representation of the cells, components andpathways found in the hepatobiliary region.

FIG. 5A is a photograph of T1-weighted magnetic resonance images of arat at various times (indicated in minutes) after injection of theGd-DTPA-bis(phenylalanine). Approximately 0.1 mmol/kg dose.

FIG. 6A is a photograph of T1-weighted magnetic resonance images atvarious times (indicated in min) obtained as in FIG. 5A and FIG. 5B forthe Gd-DTPA-bis(phenylalanine ethyl ester).

FIGS. 5B and 6B are photographic enlargements of the pre- and 0-min postinjection images of FIG. 5A and 6A, respectively.

FIG. 7 is a photograph of the T1-weighted magnetic resonance images oftwo mice side-by-side at various times (indicated in min) aftersimultaneous injection of Gd-DTPA-bis(phenylalanine) (i.e. bis acid)described in Example 8 below, at approximately 0.1 mmol/kg dose. Theimages are 2 mm thick slices in a coronal plane at the level of theheart. The heart, liver and intestines are evident.

FIG. 8 is a photograph of T1-weighted magnetic resonance images asobtained for FIG. 6 except that a different preparation ofGd-DTPA-bis(phenylalanine ethyl ester) was employed.

FIGS. 9-13 are each a graphic representation of MRI imaging in heart,lung, kidney, liver and skeletal muscle tissue, respectively, showing %enhancement versus time (min) for Gd(III)-DTPA-(3HTA)₂ and forGd(III)-DTPA-(DMPE)₂.

FIG. 14A is a photograph of T1-weighted MRI images of a rat as obtained(as indicated in min) for FIG. 5 using Gd(III)-DTPA-(3HTA)₂.

FIG. 14B is a photograph of a second coronal plane at the level of thekidneys, as shown in FIG. 14A.

FIG. 15A is a photograph of T1-weighted MRI images of a rat as obtained(as indicated in min) for FIG. 5 using Gd(III)-DTPA-(DMPE)₂.

FIG. 15B is a photograph of a second coronal plane at the level of thekidneys, as shown in FIG. 15A.

FIGS. 16-20 are each a graphic representation of MRI imaging in heart,lung, kidney, liver and skeletal muscle tissue, respectively, showing %enhancement versus time (min) for Gd(III)-DTPA-(L-PheOEt)₂ (i.e.Gd(III)-DTPA-bis(L-phenylalanine ethyl ester)) and forGd(III)DTPA-(D-PheOEt)₂ (i.e. Gd(III)-DTPA-bis(D-phenylalanine ethylester)).

FIGS. 21 (A and B) and 22 (A and B) are each T1-weighted MRIphotographic images of a rat as obtained for FIGS. 14 and 15 usingGd(III)-DTPA-(L-PheOEt)₂ (FIG. 21) or Gd(III)-DTPA-(D-PheOEt)₂ (FIG.22); except that the dose level was 0.05 mmol/kg.

On FIGS. 9 to 13 and 16 to 20 solid vertical lines within the graph areshown ending in a horizontal line. The center box of this vertical lineis the average for the observation at that point. The horizontal linesat either end of the vertical line are located at one standardderivation from the center value.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

Definitions

As used herein:

"Alkylene" refers to methylene, ethylene, propylene, and the like up toseven carbon units. This usage is equally applicable to moietiescomprising alkylene groups such as the alkyl, haloalkyl and alkoxylmoieties of the present invention.

"Amino acid" refers generally to the type of α-amino acids found inliving subjects or mammals. However, synthetic α-amino acids which arenot found in nature are also useful. Further these D- and L- amino acidsas separate chiral isomers are independently useful. Mixtures of the D-and L- isomers are also contemplated in this invention. A variety ofsuch amino acids and their derivatives are well known in the art; see,e.g., Beilsteins Handbuch der Orcianischen Chemie (Springer Verlag,Berlin) and Chemical Abstracts which provide references to publicationsdescribing the properties and preparation of such compounds.

"Metal of atomic number 21 to 31" refers to scandium, titanium,vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc andgallium respectively. Paramagnetic ions are especially preferred. Iron,manganese, nickel, chromium, cobalt are preferred.

"Metal (lanthanides) having an atomic number from 57 to 71" refers tolanthanide, cerium, praseodymium etc. to lutetium, respectively.Paramagnetic gadolinium (III) or dysprosium (III) are preferred.

"Substituted aryl" and "substituted heteroaryl" refer to moietiescontaining aromatic rings having one or more ring substituents,typically 1-3 ring substituents, that are independently selected fromrelatively non-reactive (and relatively non-toxic) groups such as alkylhaving 1-7 carbon atoms, halo, haloalkyl having 1-7 carbon atoms,hydroxyl, carboxyl, acetoxyl, alkoxyl having 1-7 carbon atoms, amino,nitro, nitroso, sulfonyl and thio; and not from relatively reactivegroups such as isothiocyanate (--N═C═S) and the like.

As is common, formulas are sometimes written on a single line withsubstituents of an atom listed (in parentheses) after the atom to whichthey are attached. Thus, for example, the formula: ##STR5## may berepresented as --(C═O)--NH--CH(R)--(C═O)--(A). Other abbreviations usedherein include, for example, --(L-PheOEt)₂ for -bis(L-phenylalanineethyl ester).

The contrast agents of this invention localize in several organ systems,e.g., in the kidney, urinary tract, and urinary bladder; in the liver,biliary tree, and intestinal lumen; and in the myocardium. Thislocalization results in increased MRI signal and image contrast. Theresulting images show both improved anatomic detail and allow thefunctional state of certain organ systems, e.g., the urinary and biliarysystems, to be ascertained.

This localization probably involves a combination of physico-chemicaland receptor-based mechanisms. For example, binding to blood componentsresults in enhancement of the blood pool and may contribute to heartenhancement. Localization in the liver may result from recognition andtransport by hepatocytes. Other mechanisms may also be involved. It maybe possible to target other organs and tissues by selective modificationof the structure of the metal chelate contrast agent.

A. AMINO-ACYL-TYPE CONTRAST AGENTS (L¹ --M)

Preparation of Amino-Acyl-Type Chelating Structures (L¹)

In one embodiment the present invention relates to polydentateamino-acyl-type chelating structures (L¹) of Formula 1, supra, wherein Ais a carbonyl-containing moiety which is an acid or ester (i.e. A is--(C═O)OR¹, and R¹ is independently selected from the group consistingof hydrogen (i.e. the acid), alkyl having 1-7 carbon atoms, cyclohexyl,phenyl, benzyl, 1-naphthyl and 2-naphthyl).

The following is a general description of the synthesis of suchchelating ligands. Specific descriptions are found in the ExperimentalSection.

In the synthesis of the compounds of Formula 1, the precursor can beDTPA-bis(anhydride) (or a similar structure, e.g. EDTA-bis(anhydride))which can be contacted with an amino acid of the structure of the knownnatural or synthetic amino acids, e.g. D, L, or mixtures thereof. Thus,the --CH₂ --(C═O)--NH--CH(R)(A) moieties may contain a chiral carbonatom (i.e. the C in --CH(R)(A)) which is a chiral center of the D or Lconfiguration and, when the contrast agent comprises a multitude of suchmoieties, the multitude of chiral centers may be either of the D or theL configuration or a mixture thereof. Generally, only single amino acidresidues are incorporated as the amino-acyl-type moieties (i.e.--NH--CH(R)(A)) in Formula 1; that is, polypeptide bonds are usually notformed.

The chelating structures of the present invention can be prepared usinga variety of natural and synthetic amino acids and their derivativesthat can be incorporated as the --NH--CH(R)(A) moiety. Such amino acidsand their derivatives are well known in the art; see, e.g., BeilsteinsHandbuch der Organischen Chemie (Springer Verlag, Berlin) and ChemicalAbstracts which provide references to publications describing theproperties and preparation of such compounds, which publications areincorporated herein by reference. Examples of substituted α-amino acidsthat can be covalently linked to, e.g., DTPA-bis(anhydride) via theirfree amino groups include esters and amides of amino acids as well astheir derivatives. Preferred α-amino acids for use in the presentinvention are phenylglycine, phenylalanine, tryptophan, tyrosine andhistidine, and substituted derivatives thereof. Preferred substitutionson the aromatic rings of these amino acids include alkyl having 1-7carbon atoms, halo, haloalkyl having 1-7 carbon atoms, hydroxyl,carboxyl, acetoxyl, alkoxyl having 1-7 carbon atoms, amino, nitro,nitroso, sulfonyl and thio. Presently, 4-t-butylphenylalanine andp-ethoxyphenylalanine are especially preferred amino acid derivativesfor use in the present invention.

References describing the properties and preparation of such amino acidderivatives can be found in numerous published sources includingBeilsteins Handbuch and Chemical Abstracts, supra. In BeilsteinsHandbuch, for example, references describing the properties andpreparation of phenylalanine, phenylglycine and tyrosine derivatives canbe found in Volume 14 of the Hauptwerke (Main Series), and Volume 14 ofthe Erganzungwerke (Supplementary series). References describing theproperties and preparation of tryptophan derivatives can be found inVolume 22 of the Hauptwerke and Volume 22 of the Erganzungwerke. Variousmethods of synthesizing amino acids and their derivatives are alsodescribed in Synthesis of Amino Acids, G. C. Barrett, Chapter 8 in"Chemistry and Biochemistry of the Amino Acids" by G. C. Barrett (ed.)(Chapman and Hall, London, 1985). A number of amino acid derivatives arealso readily available from commercial suppliers such Aldrich ChemicalCo. and Fluka Chemical Co.

With the bis(anhydride), if a limited amount (e.g. 0.5 equivalent) ofthe amino acid is used, production of the mono amino acid derivative isfavored. If two equivalents of amino acid is used, then the bis-aminoacid derivative is produced. For DTPA or higher analogs ofpolycarboxylic acids, forcing conditions, such as using a couplingreagent and a large excess of the amino acid or protected amino acid maybe required.

Any anhydrous dipolar aprotic solvent can be used for the synthesis.Dimethylformamide (DMF), dimethylacetamide, acetonitrile or the like areuseful. DMF is preferred. The reaction mixture is heated at 70 to 100°C. for between about 2-12 hr, preferably between 90 and 100° C. for 4-5hr, especially 6 hr.

The reaction mixture is cooled and the solvent is removed using aconventional rotary evaporator or its equivalent. In one aspect, thepresent invention relates to a novel preparation of the compounds ofFormula 1.

In another embodiment the present invention relates to polydentateamino-acyl-type chelating structures (L¹) of Formula 1, supra, wherein Ais a carbonyl-containing moiety which is an amide (i.e. A is--(C═O)--N(R²)(R³) wherein R² and R³ are each independently selectedfrom the same group defined for R¹, supra).

The amides and related structures (free amide, mono-substituted amide ordisubstituted amide) are produced by starting with the appropriate aminoacid amide (usually as the hydrochloride). Some purification of theamino acid may be needed.

The amino acid amide is then contacted with the correspondingdianhydride as is described above for the amino acid ester. If aless-than-equivalent amount of amino acid amide is used and at highdilution in the solvent the mono amino acid amide is favored. If astoichiometric excess of the amino acid amide is used, the diamine acidamide structure is obtained.

The amide structures are also described in Examples 12 to 22. The amidestructures are useful in MRI, because they have good contrast propertiesfor specific tissue and have a longer useful half-life in a mammaliansystem.

Preparation of the metal ion chelate complex (L¹ --M)

The general description of the preparation of chelate metal ioncomplexes is conventional in the art; see, e.g., the references citedabove.

Metal chelates are typically prepared by the reaction of a metal salt oroxide with the chelating ligand in a suitable aqueous or organic solventin the appropriate stoichiometric ratio. Elevated temperatures aresometimes required. The pH of the reaction mixture is then adjusted witha base to obtain the corresponding chelate salt. Alternatively, acid canoften be used to obtain the protonated chelate.

Preferably, in the amino-acyl-type contrast agents, at least one R or R¹group of the ligand (L¹) comprises an aromatic or heteroaromatic moiety.

The R group preferred is independently selected from --W and --K--W,wherein K is an alkyl group having 1-7carbon atoms, and W isindependently selected from the group consisting of aryl, substitutedaryl, heteroaryl and substituted heteroaryl; provided further that, if Wis substituted aryl or substituted heteroaryl, then the aromatic ringsubstituents are independently selected from relatively non-reactive(and relatively non-toxic) groups such as alkyl having 1-7 carbon atoms,halo, haloalkyl having 1-7 carbon atoms, hydroxyl, carboxyl, acetoxyl,alkoxyl having 1-7 carbon atoms, amino, nitro, nitroso, sulfonyl andthio; and not from relatively reactive groups such as isothiocyanate(--N═C═S) and the like. The K group preferred is selected from the groupconsisting of methylene, ethylene and propylene.

The more preferred R group is independently selected from the --K--Wmoieties shown below: ##STR6## and their derivatives substituted with 1to 3 groups independently selected from alkyl having 1-7 carbon atoms,halo, haloalkyl having 1-7 carbon atoms, hydroxyl, carboxyl, acetoxyl,alkoxyl having 1-7 carbon atoms, amino, nitro, nitroso, sulfonyl andthio.

Presently, the most preferred R group is --K--W wherein W is selectedfrom the group consisting of hydroxyphenyl, methoxyphenyl, ethoxyphenyland t-butylphenyl; most preferably p-ethoxyphenyl and p-t-butylphenyl.

Each R¹, R² and R³, when present, is independently selected from thegroup consisting of H (i.e. the acid), alkyl having from 1-7 carbonatoms (i.e. the mono, di, tri, etc., acid ester), and cyclic groups suchas cyclohexyl, phenyl, benzyl, 1-naphthyl and 2-naphthyl.

Preferred metal ions (M) are gadolinium (III), dysprosium (III),chromium (III), iron (II), iron (III), cobalt (III), manganese (II) andmanganese (III). Paramagnetic metal ions are preferred. Especiallypreferred are gadolinium (III) and dysprosium (III).

Presently preferred amino-acyl-type contrast agents of this inventionare gadolinium(4-t-butylphenylalanine-DTPA) andgadolinium(p-ethoxyphenylalanine-DTPA).

B. CATECHOLAMINE-TYPE CONTRAST AGENTS (L² --M)

In another aspect the present invention relates to catecholamine-typechelating structures (L²), and metal ion complexes thereof (L² --M),comprising one or more --NH--CH₂ --R⁴ moieties (wherein each R⁴ isindependently selected from the group consisting of --CH₂ -aryl, --CH₂CH₂ -aryl, --CH₂ -(substituted aryl), and --CH₂ CH₂ -(substitutedaryl)).

When a catecholamine-type compound having a --NH--CH₂ --R⁴ moiety iscontacted with the bisanhydride as described for the corresponding aminoacid ester or amide, the expected compound is obtained. When thesubstituents on the aryl group are hydroxyl, aqueous base should beavoided.

In preferred embodiments of this type, the present invention concernsthe preparation of a chelating ligand that bears one or morecatecholamine groups, making a stable chelate of this ligand with auseful metal ion, and using the chelate for diagnostic imaging orspectroscopy.

In preferred catecholamine-type compounds, R⁴ is a substituted arylwhich is selected from substituted phenyl and substituted naphthyl, andsubstituted aryl is substituted with 1 to 3 groups independentlyselected from the group consisting of hydroxyl, alkyl having 1-7 carbonatoms, alkoxyl having 1-7 carbon atoms, halo, haloalkyl, nitro, nitroso,and amino.

In more preferred catecholamine-type compounds, R⁴ is a substitutedphenyl which is substituted with two groups selected from hydroxyl,methoxyl and ethoxyl.

If the metal ion is paramagnetic, e.g., Gd(III) or Dy(III), the chelatecan produce contrast enhancement in an MRI, or cause shifts, broadening,or other changes in a magnetic resonance spectrum.

These novel agents constitute an improvement over the prior art in thatthey tend to be localized in certain types of tissue by virtue of theirresemblance to naturally occurring catecholamines and/or their redox andother physicochemical properties. Two derivatives of dopamine (also3-hydroxytyramine or "3-HTA"), namely DTPA-bis(3-hydroxytyramide) andDTPA-bis(3,4-dimethoxyphenethylamide) are particularly useful. Theseligands were reacted with Gd(III) to produce the chelates,Gd-DTPA-(3-HTA)₂ and Gd-DTPA-(3,4-DMPE)₂, respectively. These were usedas contrast agents in the MRI of rats as described in the Examples. Bothchelates demonstrated useful enhancement of heart, lungs, kidney andliver. However, the former selectively enhanced the heart.e

Determining the Efficacy of Contrast Arents of the Present Invention

MRI contrast agents have magnetic properties capable of altering imageintensity. The activity of the compounds of the present invention can beexamined directly by imaging in rodents, as is described below. However,it is also possible to obtain an initial estimate of the magneticefficacy in vitro by comparing, for example, the water proton relaxivityor the magnetic susceptibility of a compound relative to known contrastagents.

Suitable MRI contrast agents will also localize for a time in the targetcompartment, tissue or organ. Radio-labeled analogs can be used toquantify tissue concentrations. Such techniques have been described inthe art; see, e.g., K. A. Muetterties et al., "Ferrioxamine derivativesas hepatobiliary contrast agents for magnetic resonance imaging," Magn.Reson. Med. (1991), Vol. 22, pp. 88 to 100.

Hepatic uptake, for example, is generally reflected in increased biliaryexcretion and fecal elimination. Thus, hepatic uptake can be estimatedby determining the relative amounts of urinary and biliary excretion(see, e.g., Example 22, below); or by determining the relative amountsof urinary and fecal excretion (see, e.g., Example 23, below).

Finally, as described below, imaging studies in laboratory animals suchas rats, can also be used to examine the pharmacokinetics andbiodistribution of a particular contrast agent.

Magnetic Resonance Imaging

In vivo magnetic resonance imaging of organs and tissue in bothlaboratory animals and humans is now conventional and well established.

FIG. 5 is a photograph of T1-weighted magnetic resonance images of a ratobtained before, and at 0, 5, 10, 15, 25, 45 and 60 minutes after theinjection of Gd-DTPA-bis(phenylalanine) at a dose of mmol/kg bodyweight. The images are 60 mm×60 mm×3 mm thick slices in the coronalplane. The region covered extends from just above the heart to somewhatbelow the liver. Enlargements of the pre- and 0-min post images areshown in FIG. 5B. Imaging parameters are indicated along the left of theFigure and include the repetition time (3000000 microseconds), echo time(6000 microseconds), number of signal averages (4), and the image matrixsize (128×256). The increase in signal intensity, particularly in theheart and liver, are readily apparent. Increase in signal intensity ofthe intestinal lumen is particularly apparent in the 25 min and laterimages, and suggests that contrast agent has been excreted into thatorgan.

FIGS. 6A and 6B are photographs of T1-weighted magnetic resonance imagesobtained as described in FIG. 5A and 5B, except thatGd-DTPA-bis(phenylalanine ethyl ester) was used as the contrast agent.Note that this compound results in different apparent enhancement in theliver and heart as compared to that shown in FIG. 5A and 5B. Theseresults suggest that the two compounds have significantly differentbiodistributions and pharmacokinetics.

Specific experiments are described in detail below in the Examples.

Administration of Contrast Agent

Any physician can determine the best mode of administration of thecontrast agent. Generally, injection into a vein is used.

Suitable pharmaceutical compositions comprising the contrast agents ofthe present invention may include, for example, various buffers and/orstabilizers, as is well-known in the art; see, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, latest edition,Easton, Pa.

The contrast agents described herein are useful for the magneticresonance imaging of the heart, liver, biliary tree, bladder andintestine of a subject, e.g., an animal, a mammal, especially a humanbeing.

The following examples are provided to illustrate the invention--not tolimit it.

EXAMPLE 1 PREPARATION OF DTPA-BIS(PHENYLGLYCINE)

In a 50-mL round-bottom flask equipped with a magnetic stirrer and areflux condenser, and heated by an oil bath, was placed 1.10 g (3.08mmol) of DTPA-bis(anhydride) (Aldrich Chemical Co.), 0.93 g (6.15 mmol)of d,1-α-phenylglycine (Fluka Chemical Co.), and 24 mL of drydimethylformamide (Aldrich Chemical Co.). The reaction mixture washeated to 90-100° C., and held within that temperature range for 6 hr.It was then allowed to cool to room temperature, and the solvent wasremoved using a rotary evaporator. The residue was washed by triturationwith ether to yield 2 g of white solid of structure Ib (FIG. 3).

EXAMPLE 2 PREPARATION OF THE Gd(III) COMPLEXES OFDTPA-BIS(PHENYLGLYCINE)

A solution of 2 mg (30 μmol) of DTPA-bis(phenylglycine) of Example 1 in1 mL of water was treated with 14 mg (38 μmol) of GdCl₃.6H₂ O. The pH ofthe resulting mixture was adjusted to 7.0 by addition of dilute sodiumhydroxide solution. Insoluble Gd(OH)₃ was removed from the reactionmixture by filtration through a 0.22μ filter. The T1 relaxation time ofthe resulting solution (1.3 mL volume) was 7 millisecond (ms) at 0.25Tesla and 37° C.

EXAMPLE 3 MAGNETIC RESONANCE IMAGING OF A RAT USINGGd-DTPA-BIS(PHENYLGLYCINE)

A 300 g male Sprague-Dawley rat was anesthetized with an intraperitonealinjection of a mixture of ketamine and diazepam, and a catheter wasinserted into a lateral tail vein. The rat then was placed in a 5-cminside diameter (i.d.) imaging coil in the bore of a 2-Teslaimager-spectrometer system (GE CSI; General Electric Co., Fremont,Calif.). A T1-weighted spin-echo image of the animal's abdomen in thecoronal plane was then obtained (TR 315 ms; Te 15 ms; 128×256 imagematrix; NEX=4; 3 mm slice thickness). Next, 1.0 g of theGd-DTPA-bis(phenylglycine) solution described in Example 2 was injectedvia the catheter. A series of post-injection images were obtained. Theimages displayed an initial small enhancement in the liver. As thisenhancement decreased with time, increased intensity in the rat's smallintestine then was observed, indicating hepatobiliary transport of thecontrast agent. Intensity data are summarized below.

                  TABLE 1    ______________________________________    IMAGE REGION-OF-INTEREST % ENHANCEMENT    Time      Liver      Small Intestine                                       Muscle    ______________________________________    (min post-    injection)     0-3      4          14            19    15-18     7          42            2    30-33     2          32            6    ______________________________________

EXAMPLE 4 PREPARATION OF DTPA-BIS(L-PHENYLALANINE ETHYL ESTER)

L-phenylalanine ethyl ester hydrochloride, 4.6 g (20 mmol; SigmaChemical Co., St. Louis, Mo.), was dissolved in 15 mL of water andtreated with 35 mL of saturated sodium bicarbonate solution. Theresulting solution was extracted with four 10 mL portions of methylenechloride, and the organic extract was dried over anhydrous magnesiumsulfate. The dried methylene chloride solution then was filtered toremove remaining drying agent, and the filtrate was concentrated to anoil using a rotary flash evaporator. This residue was further driedunder high vacuum for several hours to yield 3.75 g of free base.

DTPA-bis(anhydride), 2.85 g (8.0 mmol), 10 mL of dimethylformamide(DMF), and 4.2 mL (24 mmol) of diisopropylethylamine (DIPEA) (SigmaChemical Co., St. Louis, Mo.) were combined in a 50 mL round-bottomflask equipped with a magnetic stirrer. The phenylalanine describedabove was dissolved in 10 mL of DMF, and the resulting solution addedvia syringe to the flask. The reaction mixture was warmed to 40° C., andthen stirred for 13 hr at ambient temperature without external heating.

At the end of the 12 hr period, the reaction mixture was concentrated invacuo to yield a viscous residue. This material was triturated with 100mL of acetone, and the volatile components of the resulting mixture wereremoved in vacuo. The solid residue was recrystallized from a mixture of125 mL of 60/40 water/ethanol. The white, crystalline product was washedwith two 25-mL portions of cold ethanol, and the washed solid was driedin vacuo at 40° C. for 1 hr to obtain 3.0 g (50% of theory).

Analytically pure product was obtained by dissolving 1 g of the abovecrystals in 75 mL of ethanol at 80-85° C., treating the resultingsolution with decolorizing charcoal, removing the latter by filtration,and cooling the filtrate in an ice bath. Seed crystals were then added,and after 45 min, 0.6 g of recrystallized solid was isolated byfiltration.

Anal: Calcd. for C₃₆ H₄₉ N₅ O₁₂ : C, 58.13; H, 6.64; and N, 9.42. Found:C, 57.75; H, 6.57; and N, 9.36.

EXAMPLE 5 PREPARATION OF DTPA-BIS(1-PHENYLALANINE BENZYL ESTER)

DTPA-bis(phenylalanine benzyl ester) was similarly prepared (accordingto Example 4) from L-phenylalanine benzyl ester p-toluene-sulfonic acidsalt, 4.28 g (10 mmol; Sigma Chemical Co., St. Louis, Mo.). Ethylacetate was used in place of ethanol for crystallization. The yield was2.6 g (75% of theory).

EXAMPLE 6 PREPARATION OF DTPA-BIS(L-PHENYLALANINE)

A solution of 1.23 g (1.42 mmol) of DTPA-bis (phenylalanine benzylester) in 15 mL of methanol was combined with 0.1 g Pd/carbon catalyst(Aldrich Chemical Co., Milwaukee, Wis.) in a 25-mL round-bottom flask.This mixture was treated with hydrogen gas at one atmosphere pressurefor 6 hr. The reaction mixture was then filtered through a bed ofdiatomaceous earth filter aid. Volatile components were removed from thefiltrate in vacuo. The yield was 0.94 g (97% of theory) of product, asomewhat hygroscopic white solid.

Anal: Calc'd. for C₃₂ H₃₁ N₅ O₁₂.2H₂ O: C, 53.10; H, 6.27; and N, 9.68.Found: C, 53.13; H. 6.24; and N, 9.32.

When examined by HPLC (see description for FIG. 8 and Example 11,below), the product was found to be about 10% bis acid, 45% bis esterand 45% mono acid monoester. This is actually the contrast agent usedfor the FIG. 6 MRI image.

EXAMPLE 7 PREPARATION OF THE GADOLINIUM (III) CHELATES OFBIS(PHENYLALANINE) AND ITS ESTERS

(a) A solution of 0.176 g (0.25 mmol) of DTPA-bis(phenylalanine) in 4 mLof water was treated with 0.093 g of GdCl₃ (Aldrich Chemical Co.,Milwaukee, Wis.). The pH of the resulting solution adjusted to 7.0 withaqueous sodium hydroxide solution. The volume was adjusted to 5.0 mLwith water, and this solution was filtered through a 0.22 micron sterilefilter into a sterile serum vial. The resulting 0.05M solution issuitable for imaging in small animals.

The T1relaxation time at 0.25 Tesla magnetic field strength and 37° C.of a fivefold dilution of the above solution was 21 ms.

(b) The DTPA-bis(phenylalanine) mono and bis esters were prepared in asimilar fashion.

EXAMPLE 8 MAGNETIC RESONANCE IMAGING OF A RAT USINGGd-DTPA-BIS(PHENYLALANINE)

A 300-g male Sprague-Dawley rat was anesthetized with an intraperitonealinjection of a mixture of ketamine and diazepam, and a catheter wasinserted into a lateral tail vein. The rat was then placed in a 5-cminside diameter (i.d.) imaging coil in the bore of a 2-Teslaimager-spectrometer system (GE CSI; General Electric Co., Fremont,Calif.). A T1-weighted spine-cho image of the animal's abdomen in thecoronal plane was then obtained (TR 300 ms; Te 6 ms; 128-256 imagematrix; NEX=4; 3 mm slice thickness). Next, 0.6 g of theGd-DTPA-bis(phenylalanine) solution described in Example 7 was injectedvia the catheter. A series of post-injection images were obtained. Theimages displayed an initial enhancement in the liver and heart. As thisenhancement decreased somewhat with time, increased intensity in therat's small intestine then was observed, indicating hepatobiliarytransport of the contrast agent. Intensity data are summarized below.The intensity values show some fluctuations due to breathing motion andother small artifacts. See also, FIGS. 5A and 5B.

                  TABLE 2    ______________________________________    IMAGE REGION-OF-INTEREST % ENHANCEMENT    Time      Liver      Heart         Muscle    ______________________________________    (min post-    injection)     0-3      51         42            14     5-8      60         27            3    15-18     47         25            12    25-38     50         22            12    45-48     34         9             10    60-63     33         12            2    ______________________________________

EXAMPLE 9 MAGNETIC RESONANCE IMAGING OF A RAT USINGGd-DTPA-BIS(PHENYLALANINE ETHYL ESTER)

The imaging was carried out analogously to Example 8. Intensity data aresummarized below. See also, FIGS. 6A and 6B.

                  TABLE 3    ______________________________________    IMAGE REGION-OF-INTEREST % ENHANCEMENT                                    Skeletal    Type      Liver      Heart      Muscle    ______________________________________    (min post-    injection)     0-3      70         86         50     5-8      109        46         30    15-18     113        66         31    25-38     99         48         26    45-48     71         49         16    60-63     44         25         16    ______________________________________

EXAMPLE 10 MAGNETIC RESONANCE IMAGING OF MICE USINGGd-DTPA-BIS(PHENYLALANINE) (BIS PHE ACID about 100%)

Two male BALB mice were imaged side by side in the same apparatus andusing the same conditions as found in Example 8, except that the slicethickness was 2 mm. See FIG. 7 and accompanying description. Over theillustrated time course from 0 min to 2.5 hr, the contrast agent can beseen to localize first in the liver (e.g., at 2 min), then in the gallbladder (at 90 min, for example), and then in the intestinal lumen(2-2.5 hr). It can also be seen in the urinary bladder.

EXAMPLE 11 COMPARATIVE MRI DATA IN MICE

FIG. 8 is a photograph of T1-weighted magnetic resonance images obtainedas in FIG. 6, except that a different preparation ofGd-DTPA-bis(phenylalanine ethyl ester) (sometimes abbreviatedGd-DTPA-(PheOEt)₂) was used.

When examined by HPLC 4.6×150 mm PRP-1 column; mobile phase 25 mMammonium formate in water (Solvent A) and 50/50 (v/v) acetonitrile/water(Solvent B), programmed from 10% B to 95% B over 15 min, then holding at95% B; flow rate 1 ml/min!; UV and/or radioisotope detector, thematerial used as a contrast agent in FIG. 6 was found to have partiallyhydrolyzed to a mixture of Gd-DTPA-(PheOEt)₂ (i.e. bis ester) (ca. 45%);Gd-DTPA-(PheOEt)(Phe) (i.e. mono ester, mono acid) (ca. 45%); andGd-DTPA-(Phe)₂ (i.e. bis acid) (ca. 10%).

Freshly prepared material, whose pH was carefully adjusted toneutrality, and which was stored in the cold, was determined to be about90% Gd-DTPA-(PheOEt)₂, the remainder being mostly Gd-DTPA(PheOEt) (Phe).

The more pure preparation gave heart and liver enhancement (74% and163%, respectively) as shown in FIG. 6 (84% and 53%, respectively).Thus, the degree of liver enhancement was greater by a factor of aboutthree.

These results suggest that esterified DTPA-amino acid chelates may beparticularly advantageous for higher contrast enhancement.

EXAMPLE 12 PREPARATION OF DTPA-BIS(D-PHENYLALANINE ETHYL ESTER)

DTPA-bis(D-phenylalanine ethyl ester) was prepared analogously to thecorresponding L-isomer from D-phenylalanine ethyl ester andDTPA-bis(anhydride) (Example 4). The yield was 65%.

Anal. Calcd for C₃₆ H₄₉ N₅ O₁₂ : C, 58.13; H, 6.64; and N, 9.42. Found:C, 57.87; H, 6.55; N, 9.48.

EXAMPLE 13 PREPARATION OF DTPA-BIS(PHENYLALANINE METHYLAMIDE)

A suspension of 2.07 g (10.43 mmol) of L-phenylalanine methyl amidehydrochloride in ethyl acetate (75 mL) was treated with a saturatedaqueous solution of sodium carbonate (20 mL). The resulting solution wasextracted with ethyl acetate (2×75 mL), and the combined organicextracts were dried over anhydrous sodium sulfate. The drying agent wasremoved by filtration, and the filtrate was concentrated to an oil usinga rotary evaporator. This residue was further dried over P₂ O₅ underhigh vacuum overnight to yield 1.72 g of the amine as a white solid.

A solution of the dried amine in anhydrous pyridine (15 mL) was combinedwith DTPA-bis(anhydride) (1.80 g, 5.04 mmol) under argon. The reactionmixture was heated at reflux in an oil bath (95° C.) for 60 min. Themixture was allowed to cool to room temperature (0.5 hr) and wasconcentrated in vacuo to yield a viscous residue. This material wasdissolved in 100 mL of water, and the water then was evaporated in vacuoto yield a yellow oil. The oil was dissolved in a minimum amount of asolution of water in methanol (20% v/v) and treated with acetonitrileuntil a small amount of precipitate was observed. The precipitate wasremoved by filtration (0.45 μm membrane filter), and the filtrate wasconcentrated under reduced pressure. This procedure was repeated twicemore, discarding the precipitate each time. Finally, the residueobtained by evaporation of the solvent was dissolved in a solution ofwater in methanol (10 mL, 20% v/v), and the desired product wasprecipitated by addition of a minimum amount of acetonitrile. Theresulting white suspension was cooled in a freezer (-20° C.) overnight,and the solvent then was removed by decantation. The product was driedunder high vacuum (0.05 torr, 48 hr) over P₂ O₅ and NaOH to afford 1.38g (39%) of an analytically pure white solid.

Anal. Calcd. for C₃₄ H₄₇ N₇ O₁₀.0.5 H₂ : C, 56.50; H, 6.69; N, 13.57.Found: C, 56.34; H, 6.61; N, 13.66.

EXAMPLE 14 PREPARATION OF DTPA-BIS(PHENYLALANINE AMIDE) ANDDTPA-BIS(PHENYLALANINE DIMETHYLAMIDE)

The title compounds (i.e. DTPA-bis(phenylalanine amide) andDTPA-bis(phenylalanine dimethylamide) were prepared analogously to thedimethylamide compound of Example 13 from DTPA-bis(anhydride) andL-phenylalanine amide hydrochloride and L-phenylalanine dimethylamide;in 20% and 59% yields, respectively.

Calcd. for the amide C₃₂ H₄₃ N₇ O₁₀.2H₂ O: C,53.25; H,6.57; N,13.58.Found: C,53.43; H,6.30; N,13.59.

Calcd for the dimethylamide C₃₆ H₅₁ N₇ O₁₀ : C,56.90; H,7.03; andN,12.90. Found: C,56.82; H,6.74; N,12.76.

EXAMPLE 15 PREPARATION OF DTPA-BIS(3-HYDROXYTYRAMIDE) ("DTPA-(3-HTA)₂ ")

DTPA-bis(anhydride) (3.57 g; 1.00 mmol; Aldrich Chemical Co., Milwaukee,Wis.) was suspended in 25 mL of anhydrous dimethylformamide (DMF;Aldrich) and treated with dopamine (3.78 g; 2.00 mmol; Fluka-USA,Ronkonkoma, N.Y.) and di-isopropylethylamine (5.2 g; 4.0 mmol; AldrichChemical Co.) This mixture then was heated briefly to 100° C. andsonicated for several minutes to dissolve the bulk of the solid. Afterstirring for 4-6 hr at 50-60° /cm, a deep yellow solution was produced.The reaction mixture then was allowed to cool to room temperature.

After stirring at ambient temperature overnight, the reaction mixturewas concentrated on a rotary evaporator at 50° C. to a volume of about10 mL. The odor of di-isopropylethylamine was absent at this point.Water (25 mL) was added, and the resulting solution was washed twicewith 20 mL portions of ethyl ether to remove the remaining DMF. Thewater then was removed in vacuo to yield a beige paste. This materialwas suspended in 10-20 mL of absolute ethanol and dried by azeotropicdistillation of aqueous ethanol in vacuo. The crude product (7 g; 97% oftheory) was a gritty, off-white, hygroscopic solid.

An analytical pure sample (1.76 g; 26% of theory) was isolated bypreparative high-pressure liquid chromatography (HPLC) using a 4.6×150mm Microsorb C-18 reversed phase column (Rainin Instrument Co.,Emeryville Calif.). The mobile phase (1 mL/min flow rate) was a lineargradient from 5 to 50% acetonitrile in water over 12 min An acidic pHwas maintained by the presence of 0.1% v/v trifluoroacetic acid in bothcomponents of the mobile phase. A UV detector measuring absorbance at276 nm was used. The retention time of the product was 13.5 min underthese conditions. ¹ H NMR spectrum: δ6.75, m, 6H; δ3.81-2.62, 26H,aliphatic H, not further assigned.

Liquid secondary ion mass spectrum (LSIMS) M--H!=662 (theory 662).

EXAMPLE 16 PREPARATION OF DTPA-BIS(3,4-DIMETHOXYPHENETHYLAMIDE)("DTPA-(3,4-DMPE)₂ ")

Equimolar amounts of DTPA-bis(anhydride) and 3,4-dimethoxyphenethylamine(Aldrich Chemical Co.) were contacted as above in Example 15 to givecrude product in ca. 100% yield. This material was purified bypreparative HPLC to produce 1.23 g (17%) of the title compound.

¹ H NMR spectrum: δ6.80, m, 6H; δ3.82, s, 6H, CH₃ O--; δ3.80, s, 6H; CH₃O--; δ3.45-2.76, 26H, aliphatic H not further assigned.

LSIMS mass spectrum: M--H!-=718 (theory 718).

EXAMPLE 17 PREPARATION OF GD-DTPA-(3-HTA)₂

Solutions of Gd-DTPA-(3-HTA)₂ for imaging experiments were prepared byreacting DTPA-(3-HTA)₂ in aqueous solution with a stoichiometric amountof GdCl₃ dissolved in water. After about 90% of the GdCl₃ had beenadded, the pH of the reaction mixture was adjusted to between 5 and 6with aqueous NaOH solution. Xylenol orange indicator (1 drop of a 1mg/mL aqueous solution) then was added, and GdCl₃ solution was addeddropwise until the indicator changed from yellow to violet (at pH <6).The pH then was adjusted to between 7 and 8 with aqueous NaOH and, if.necessary, aqueous HCl solution. The reaction mixture was passed througha 0.22 μm sterile filter into a sterile serum vial. The finalconcentration ranged from 0.02 to 0.5M, depending upon the initialconcentrations of the reactants and the volumes of base and acid addedfor pH adjustment.

A sample of product for mass spectral analysis was obtained by HPLC(4.6×150 PRP-1 column; 1 mL/min flow rate; 5-45% over 15 minacetonitrile-in-water gradient). The LSIMS M+H!⁺ parent ion peaks wereobserved from 815-824, with the maximum intensity at 819. The ratios ofpeak intensities were those predicted by theory C₃₀ H₃₈ GdN₅ O₁₂.

EXAMPLE 18 PREPARATION OF GD-DTPA-(3,4-DMPE)₂

This chelate was prepared analogously to Gd-DTPA-(3-HTA)₂, as in Example17 above, from DTPA-(3,4-DMPE)₂ and GdCl₃.

EXAMPLE 19 IN VIVO MAGNETIC RESONANCE IMAGING USING GD-DTPA-(3-HTA)₂ andGD-DTPA-(3,4-DMPE)₂

Magnetic resonance imaging was carried out using a CSI 2-Tesla imager(GE, Inc., Fremont, Calif.) equipped with a 5-cm diameterdistributed-capacitance imaging coil. A T1-weighted (TR 300/TE 6; NEX 4)spin-echo sequence was used. The image matrix was 128×256, the slicethickness was 3 mm, and the field of view was 90 mm. anterior (heartlevel) and posterior (kidney level) coronal image planes were used.

Sprague-Dawley rats (250-350 g; n=4 for each contrast agent) wereanesthetized with ketamine (90 mg/kg) and diazepam (10 mg/kg) and fittedwith an intravenous catheter in a lateral tail vein. Anesthesia wasmaintained during imaging using pentobarbital delivered via anintraperitoneal catheter.

The anesthetized animal was placed in the imaging coil and secured withtape. The coil containing the animal then was placed in the magnet bore,and the magnetic field was shimmed. Pre-contrast images were obtained.The contrast agent (100 μmol/kg) then was injected via the tail-veincatheter, and additional images were obtained at various intervals forup to 90 min post injection.

Contrast agent enhancement was determined by measuring the mean signalintensity (SI) in operator-designated regions of interest (ROI). Thesewere normalized to the pre-injection value for each ROI according to thefollowing formula:

% Enhancement=100×(SI_(post) -SI_(prc))/SI_(prc) function of time inheart, lung, kidney, liver, and skeletal muscle, FIGS. 9-13respectively, for each of the contrast agents are illustrated,Gd-DTPA-(3-HTA)₂ also tended to produce higher lung enhancement(186%±51% vs. 141%±4%). However, the differences between the effectsproduced by the two agents was smaller than in heart (cf. FIGS. 9 and10).

There was no significant difference in kidney enhancement (FIG. 11).Both agents produced ca. 175% enhancement 5 min after injection. Thelevel of enhancement fell slowly over 70 min to about 100%.

About 50% enhancement was produced in the liver by both agentsimmediately post-injection (FIG. 12). Additionally, the time course ofenhancement was very similar for both agents, with the enhancement levelfalling to about 30% during the first 20 min post injection.

Skeletal muscle displayed peak enhancement of about 40% immediatelypost-injection. The enhancement-time curves for both agents were almostidentical; each fell almost to pre-injection levels over 80 min (FIG.13).

Representative images using each agent are shown in FIGS. 14A and 14Band 15A and 15B as MRI photographic images.

EXAMPLE 20 IN VIVO MAGNETIC RESONANCE IMAGING USING

GD-DTPA-(L-PheOEt)₂ and GD-DTPA-(D-PheOEt)₂

The magnetic resonance imaging characteristics of the two contrastagents Gd-DTPA-(L-PheOEt)₂ and Gd-DTPA-(D-PheOEt)₂ were compared as inthe previous Example using groups of 4 and 5 animals, respectively.FIGS. 16 to 20 illustrate the contrast enhancement versus time behaviorfor each agent in heart, lung, kidney, liver and skeletal muscle,respectively.

Representative images using each agent are shown in FIGS. 21A and 21Band 22AA and 22B as MRI photographic images.

EXAMPLE 21 HYDROLYSIS OF GD-DTPA-(L-PheOEt)₂ AND Gd-DTPA-(D-PheOEt)₂ INpH 7.4 BUFFER AND RAT PLASMA

The rates of hydrolysis of the esters in rat plasma or pH 7.4 HEPESbuffer were determined by addition of 10% by volume of Gd-153radiolabeled 0.025M chelate solution and incubation at 0 or 25° C.Aliquots were withdrawn at various time intervals and examined by HPLCPRP-1 column; water-acetonitrile gradient; 25 mM ammonium formate, pH 7mobile phase!.

The hydrolysis of either the LL- or DD-bis(ester) enantiomers to thecorresponding mono(ester)--mono(acid) and thence to the bis(acid) inaqueous HEPES buffer at pH 7.4 and 25° C. is very slow, with half-timesfor each step of the order of days.

However, the LL-bis(ester) is very rapidly hydrolyzed in rat plasma tothe mono(acid)-mono(ester) (see below). The latter compound is much moreresistant to hydrolysis of the remaining ester, with essentially noreaction being observed within 2 hr at 25° C.

In contrast, the DD-bis(ester) is resistant to even the first step ofester hydrolysis under these conditions (see below).

    ______________________________________    t.sub.1/2  of Ester Hydrolysis in Rat Plasma                    @ 0° C.                                @ 25° C.    ______________________________________    Gd-DTPA-(L-PheOEt).sub.2                    33 min      0.3 min    Gd-DTPA-(D-PheOEt).sub.2                    None Detected                                None Detected    ______________________________________

The relative stability of the bis(esters) toward hydrolysis in aqueoussolution versus plasma suggest that the plasma reaction isenzyme-catalyzed. Furthermore, mono(acid)-mono(ester) is evidently amuch poorer substrate, as its rate of hydrolysis is much slower. Thismay be due to the change in net charge (from 0 to 1) of the chelateand/or to a change in conformation of the molecule due to coordinationof the Gd by the free phenylalanine carboxylate group.

Changing the stereochemistry of the amino acid portion of the chelate tothe unnatural D-enantiomer caused the rate of ester hydrolysis in plasmato greatly decrease.

EXAMPLE 22 DETERMINATION OF RELATIVE AMOUNTS OF URINARY AND BILIARYEXCRETION

Male Sprague-Dawley rats were anesthetized with an intraperitonealinjection of mixture of ketamine (90 mg/kg) and diazepam (2 mg/kg), andfitted with a 23-gauge cannula placed in a lateral tail vein. Next, amidline incision and small lateral cut over the bile duct were made, andthe bile duct was exposed. Two loose ties were placed proximally on thebile duct. A small nick was made distally, and the bile duct wascannulated with a 15-cm length of PE-10 polyethylene tubing, which wassecured with the two ties.

A second piece of tubing was placed in the urinary bladder and securedwith a purse-string suture. The flap of the abdominal wall was closed,and the incision was covered with gauze.

Heparinized (1 unit/mL) saline was infused at a rate of 0.075 mL/min viathe iv catheter. After a 15 min stabilization period, the infusion wasinterrupted long enough to deliver a bolus dose (0.1 mmol/kg) of Gd-153labeled contrast agent, and then resumed. Samples of bile and urine werecollected in tared tubes at regular intervals before and after injectionof radiolabeled agent. The net weights of these samples were determined.The amount of Gd-153 present in each sample was determined by countingin a chamber gamma counter. The raw counts were corrected for backgroundand normalized to the total amount of Gd-153 injected.

The Table below summarizes the results (cumulative 1 hr excretion;average of 3 animals) obtained for some of the agents described in theprior Examples:

    ______________________________________    One-Hour Cumulative Excretion                       Biliary  Urinary    ______________________________________    Gd-DTPA-(L-Phe).sub.2                        9.3 ± 1.3                                66.5 ± 8.7    Gd-DTPA-(L-PheOEt).sub.2                       30.5 ± 7.4                                46.9 ± 8.0    Gd-DTPA-(D-PheOEt).sub.2                       51.3 ± 5.1                                39.2 ± 5.5    Gd-DTPA-(L-PheNHCH.sub.3).sub.2                        3.5 ± 0.4                                70.9 ± 6.5    ______________________________________

Alternatively, the radio-labeled contrast agent can be delivered withoutany unlabeled carrier. In this case, essentially all of the paramagneticmetal ion is present as the gamma-emitting isotope, and the absolutedose of chelate is approximately 1×10⁻⁵ mmol/kg. A significantly higherbiliary excretion level at a tracer dose, compared to that of acarrier-added dose, suggests that the latter may have exceeded thecapacity of the hepatobiliary transport system.

EXAMPLE 23 DETERMINATION OF RELATIVE AMOUNTS OF URINARY AND FECALELIMINATION

A Sprague-Dawley rat was anesthetized with an intraperitoneal injectionof a mixture of ketamine (90 mg/kg) and diazepam (2 mg/kg). A 23-gaugecannula placed in a lateral tail vein was used to deliver a bolusintravenous dose (0.1 mmol/kg) of chelate labeled with Gd-153 (ca. 0.03mCi/mmol). After injection, the rat was placed in a chamber gammacounter to determine the count rate of the injected dose. The animalthen was housed in a metabolic cage where urine and feces could beseparately collected. The animal itself, its urine, and its feces werecounted at various time points up to five days post injection.Typically, a group of four animals was used for each compound tested.The mean values for residual activity in the body, and cumulative fecaland urinary activities, expressed as a per cent of the injected dose,were determined.

As in Example 22, tracer versus carrier-added doses were sometimescompared to check for possible saturation of biliary transport at thehigher dose.

Gd-DTPA-(L-PheOH)₂ at tracer dose gave values at 2.1%±0.2%, 80.7%±8.4%,and 14.1%±1.7% for residual, urinary, and fecal activity, respectively.The corresponding values for a 0.1 mmol/kg dose were 1.8%±0.1%,81.1%±9.0%, and 12.0%±3.8%.

EXAMPLE 24 PREPARATION OF VARIOUS SUBSTITUTED PHENYLALANINE ANDSUBSTITUTED TYROSINE DERIVATIVES

(a) (L)-p-t-butylphenylalanine ((L)-p-t-butyl-PHE) was prepared via aFriedel-Crafts reaction employing (L)-phenylalanine and t-butyl alcohol.To a homogeneous solution of (L)-phenylalanine (33 g, 0.2 mol, dissolvedin 55 mL of concentrated sulfuric acid) was added t-butyl alcohol (50mL, 0.53 mol) at ambient temperature. The reaction temperature was keptat 30° C. or below during the addition of the alcohol. After stirringovernight, the reaction mixture was treated with water (100 mL) andextracted with ethyl acetate (100 mL) twice. The aqueous layer wascooled in an ice-water bath and the pH of the solution adjusted to 5.5by the addition of 6.6M NaOH, causing precipitation of the amino acid.The solid, obtained after removal of the solvent from the filtrate, wasdried in vacuo to obtain the expected product (13.9 g),(L)-p-t-butylphenylalanine, in 31% yield. Anal. Calcd. C₁₃ H₁₉ NO₂.0.75H₂ O: C, 66.50; H, 8.80; N, 5.97. Found: C, 66.46; H, 8.64; N, 5.81.

Methods for the preparation of (L)-p-t-butylphenylalanine have also beenreported in the literature (see, e.g., Miyaka, A., et al., J. TakedaRes. Lab., 43:53-76, 1984).

(b) (L)-p-ethoxyphenylalanine ((L)-p-EtO-PHE) was prepared fromN-t-boc-protected tyrosine essentially following a reported procedure(Mndzhoyan, O. L., et al., Khim. Farm. Zh., 5:4-7,1971). ToN-t-boc-tyrosine (56.3 g, 0.2 mol) dissolved in DMSO (400 mL) and 20%aqueous sodium hydroxide (80 mL) was added diethyl sulfate (33.9 g, 0.22mol) dropwise. The reaction temperature was maintained at 40-500° C. forabout 3 hours. To the reaction mixture was added 6.0M hydrochloric acid(200 mL), and the mixture was kept at 50°-60° C. for about 3 hours. Themixture was then cooled down to ambient temperature, and treated withconcentrated ammonium hydroxide until the pH of the resulting solutionwas adjusted to about pH 7. The precipitate was collected and dried invacuo. The expected product, (L)-p-ethoxyphenylalanine, was obtained in90% yield. The physicochemical properties of the (L)-amino acid were inagreement with reported literature values.

EXAMPLE 25 PREPARATION OF VARIOUS DTPA-BIS(AMINO ACID DERIVATIVES) FROMSYBSTITUTED PHENYLALANINE AND SUBSTITUTED TYROSINE

The amino acid derivatives of Example 24 were used to prepare a varietyof corresponding DTPA-bis(amino acid derivatives). In particular, thevarious DTPA-bis(amino acid) derivatives were prepared from thereactions of DTPA-bis-anhydride (1 equivalent) with the variousp-substituted amino acids (2.1 equivalents) in pyridine at 40-60 degreesCelsius for 4-6 hours under a nitrogen atmosphere. The brown residue,after removal of the pyridine, was purified by anion exchange columnchromatography using an AG1x2 (formate) column (Bio-Rad, Richmond,Calif.). The p-substituted ligands were eluted out with 2.0M aqueousformic acid.

EXAMPLE 26 PREPARATION OF GD(III) COMPLEXES OF VARIOUS DTPA-BIS(AMINOACID DERIVATIVES)

The DTPA-bis(amino acid derivatives) of Example 25 were used to preparea variety of corresponding Gd(III) complexes substantially as describedin Example 2. The gadolinium chelates were purified by reverse phasecolumn chromatography using a CHP20P column (YMC, Wilmington, N.C.).

EXAMPLE 27 DETERMINATION OF BILIARY AND/OR FECAL EXCRETION OF THEGD(III) COMPLEXES OF VARIOUS DTPA-BIS(AMINO ACID DERIVATIVES)

The Gd(III) complexes of Example 26 were introduced into rats andurinary and biliary and/or fecal excretion was determined substantiallyas described in Examples 22-13. The excretion data indicated that all ofthese contrast agents underwent hepatobiliary and renal uptake andsuggested that they would be useful in MRI.

Gd-DTPA-(L-EtO-PheOH)₂ at tracer dose gave values of 1.6%±0.7%,61.7%±7.6%, and 43.0%±13.9% for residual, urinary and fecal activity,respectively. The corresponding values for a 0.1 mmol/kg dose were1.8%±0.8%, 71.4%±4.4%, and 29.6%±3.9%. One-hour cumulative urinary andbiliary excretion levels after injection of a tracer dose were45.0%±2.1% and 26.4%±8.4%, respectively. The corresponding values for adose of 0.1mmol/kg were 55.4%±2.0% and 23.6%±2.2%.

Gd-DTPA-(L-t-Bu-PheOH)₂ at tracer dose gave values of 0.9%±0.2%,33.2%±10.6%, and 71.0%±3.5% for residual, urinary, and fecal activity,respectively. One-hour cumulative urinary and biliary excretion levelsafter injection of a tracer dose were 18.8%±1.4% and 69.3%±2.0%,respectively.

EXAMPLE 28 MAGNETIC RESONANCE IMAGING USING GD(III) COMPLEXES OF VARIOUSDTPA-BIS(AMINO ACID DERIVATIVES)

The Gd(III) complexes of Example 26 were used as contrast agents inmagnetic resonance imaging of a rat, substantially as described inExample 3. The images indicated that all of these contrast agentsunderwent hepatobiliary and renal uptake and were useful in MRI.

Gd-DTPA-(L-EtO-PheOH)₂ at 0.1 mmol/kg dose resulted in the followingmean (n=4) contrast enhancement values (as defined in Example 19) in theliver: at 5 minutes, 86%±15%; at 15 minutes, 87%±7%; and at 70 minutes,52%±3%. The corresponding values for the renal cortex were: at 5minutes, 204%±49%; at 15 minutes, 137%±9%; and at 70 minutes, 54%±3%.The following values were obtained for skeletal muscle: at 5 minutes,32%±3%; at 15 minutes, 13%±9%; and at 70 minutes, 6%±6 %.

Gd-DTPA-(L-t-Bu-PheOH)₂ at 0.1 mmol/kg dose resulted in the followingcontrast enhancement (per cent change in signal intensity) values in theliver: at 5 minutes, 104%±5%; at 15 minutes, 80%±10%; and at 70 minutes,30%±39%. The corresponding values for the renal cortex were: at 5minutes, 126%±51%; at 15 minutes, 94%±18%; and at 70 minutes, 52 %±21%.The following values were obtained for skeletal muscle: at 5 minutes,52%±18%; at 15 minutes, 54%±17% and at 60 minutes, 21%±22%.

While some embodiments of the invention have been shown and describedherein, it will become apparent to those skilled in the art that variousmodifications and changes can be made in the amino acid containinghepatobiliary or cardiac contrast agents or their use in magneticresonance imaging of the torso or abdomen of a mammal without departingfrom the spirit and scope of the present invention. All suchmodifications and changes coming within the scope of the appended claimsare intended to be carried out thereby.

We claim:
 1. A method of preparing a polydentate amino-acyl-typechelator (L¹) which method comprises:(a) contacting an anhydride of thefollowing formula: ##STR7## wherein D and E are CH₂ (C═O)OR¹, with anamino acid of the structure H₂ N--CH(R)--(C═O)OH, an ester of thestructure H₂ NCH(R)--(C═O)OR¹ or an amide of the structure H₂N--CH(R)--(C═O)--N(R²)(R³); wherein each R is independently selectedfrom the group consisting of --K, --W and --K--W, wherein each K is analkyl group having 1-7 carbon atoms, and each W is independentlyselected from the group consisting of aryl, substituted aryl, heteroaryland substituted heteroaryl; R¹, R² and R³ are independently selectedfrom hydrogen (i.e. the acid), alkyl having 1-7 carbon atoms,cyclohexyl, phenyl, benzyl, and 1- or 2-naphthyl, and m is selected from0, 1, 2 or 3, and n is selected from 0 or 1; and (b) removing thesolvent and recovering a polydentate amino-acyl-type chelator of thefollowing formula: ##STR8## wherein Q, J and Z are each independentlyselected from the group consisting of --CH₂ (C═O)OR¹ and --CH₂--(C═O)--NH--CH(P)(A), and D and E are as defined above; wherein each Ris independently selected from the group consisting of --K, --W and--K--W, as defined above; wherein the catom in --CH(R)(A) of each of the--CH₂ --(C═O)--NH--CH(R)(A) moieties is a chiral center of the D or Lconfiguration and, when the contrast agent comprises a multitude of suchmoieties, the multitude of chiral centers are either of the D or the Lconfiguration or a mixture thereof; and each A is a carbonyl-containingmoiety independently selected from the group consisting of --(C═O)OR¹and --(C═O)--N(R²)(R³), wherein R¹, R² and R³ are independently selectedfrom the group consisting of hydrogen (i.e. the acid), alkyl having 1-7carbon atoms, cyclohexyl, phenyl, benzyl, 1-naphthyl and 2-naphthyl; andm and n are as defined above.
 2. The method of claim 1, wherein theanhydride is contacted with an amino acid of the structure:H₂N--CH(R)--(C═O)OH.
 3. The method of claim 1, wherein the anhydride iscontacted with an ester of the structure:H₂ N--CH(R)--(C═O)OR¹.
 4. Themethod of claim 1 wherein the anhydride is contacted with an amide ofthe structure:H₂ N--CH(R)--(C═O)--N(R²)(R³).
 5. The method of claim 1,wherein the contacting step (a) is conducted in an anhydrous dipolaraprotic solvent at a temperature of between about 50 and 150° C.
 6. Themethod of claim 5, wherein the dipolar aprotic solvent is independentlyselected from dimethylsulfoxide or mixtures thereof.
 7. The method ofclaim 1, wherein m is 1 and n is
 0. 8. The method of claim 1, wherein atleast one R is selected from the group consisting of --W and --K--W. 9.The method of claim 1 wherein at least one R comprises an aryl groupsubstituted with 1 to 3 groups independently selected from alkyl having1-7 carbon atoms, halo, haloalkyl having 1-7 carbon atoms, hydroxyl,carboxyl, acetoxyl, alkoxyl having 1-7 carbon atoms, amino, nitro,nitroso, sulfonyl and thio.
 10. The method of claim 8 wherein aryl isindependently selected from phenyl and naphthyl, and wherein substitutedaryl is independently selected from substituted phenyl and substitutednaphthyl.
 11. The method of claim 8, wherein at least one R is --K--W,wherein K is selected from the group consisting of methylene, ethyleneand propylene, and W is substituted phenyl.
 12. The method of claim 11,wherein substituted phenyl is substituted with 1 to 3 groupsindependently selected from hydroxyl, alkyl having 1 to 5 carbon atomsand alkoxyl having 1 to 5 carbon atoms.
 13. The method of claim 12,wherein K is methylene and substituted phenyl is selected from the groupconsisting of hydroxyphenyl, methoxyphenyl, ethoxyphenyl andt-butylphenyl.
 14. The method of claim 13, wherein substituted phenyl isselected from the group consisting of p-ethoxyphenyl andp-tert-butylphenyl.
 15. The method of claim 8, wherein at least one Rcomprises a heteroaryl or substituted heteroaryl,wherein heteroaryl isselected from the group consisting of indolyl and imidazolyl andsubstituted heteroaryl is selected from the group consisting ofsubstituted indolyl and substituted imidazolyl.
 16. The method of claim13, wherein m is 1, n is 0, R is --K--W, K is methylene and W isp-ethoxyphenyl.
 17. The method of claim 13, wherein m is 1, n is 0, R is--K--W, K is methylene and W is p-tert-butylphenyl.
 18. The method ofclaim 5, wherein the dipolar aprotic solvent is independently selectedfrom dimethylsulfoxide or mixtures thereof andthe heating temperature isbetween about 90 and 100° C. and the time is between about 2 and 10hours.